In scanning microscopy, a sample is illuminated with a light beam in order to observe the detection light emitted, as reflected or fluorescent light, from the sample. The focus of an illuminating light beam is moved in a specimen plane by means of a controllable beam deflection device, generally by tilting two mirrors, the deflection axes usually being perpendicular to one another so that one mirror deflects in the X direction and the other in the Y direction. Tilting of the mirrors is brought about, for example, by means of galvanometer positioning elements. The power level of the detection light coming from the specimen is measured as a function of the position of the scanning beam. The positioning elements are usually equipped with sensors to ascertain the present mirror position.
In confocal scanning microscopy specifically, a specimen is scanned in three dimensions with the focus of a light beam.
A confocal scanning microscope generally comprises a light source, a focusing optical system with which the light of the source is focused onto an aperture (called the “excitation pinhole”), a beam splitter, a beam deflection device for beam control, a microscope optical system, a detection pinhole, and the detectors for detecting the detected or fluorescent light. The illuminating light is coupled in via a beam splitter. The fluorescent or reflected light coming from the specimen travels back through the beam deflection device to the beam splitter, passes through it, and is then focused onto the detection pinhole behind which the detectors are located. Detection light that does not derive directly from the focus region takes a different light path and does not pass through the detection pinhole, so that a point datum is obtained which results, by sequential scanning of the specimen, in a three-dimensional image. A three-dimensional image is usually achieved by acquiring image data in layers, the path of the scanning light beam on or in the specimen ideally describing a meander (scanning one line in the X direction at a constant Y position, then stopping the X scan and slewing by Y displacement to the next line to be scanned, then scanning that line in the negative X direction at constant Y position, etc.). To make possible acquisition of image data in layers, the sample stage or the objective is shifted after a layer is scanned, and the next layer to be scanned is thus brought into the focal plane of the objective.
Spectral detectors are often used for detection; these can be embodied, for example, as multi-band detectors as disclosed e.g. by German Application DE 198 03 151.3 A1.
Coherent anti-Stokes Raman scattering (CARS) microscopy is a technique that is becoming increasingly important. One great advantage is that samples do not need to be labeled with dyes. In addition, living cells can be investigated.
As compared to conventional Raman microscopy and known confocal Raman microscopy, in CARS microscopy a higher detection light yield can be obtained, troublesome secondary effects can be better suppressed, and detection light can be more easily separated from the illuminating light. Conventional confocal Raman spectroscopy requires a detection pinhole in order to achieve good axial resolution, as well as a high-resolution spectrometer. CARS, on the other hand, is a nonlinear optical process (four-wave mixing process). Similarly to the situation with multi-photon microscopy, in which two or more photons are absorbed simultaneously because the probability that multiple photons in correct phase will meet simultaneously is greatest at the focus due to the higher photon density, no detection pinhole is required. Without a detection pinhole, the same axial resolution as in multi-photon microscopy is achieved. Two lasers emitting light of different wavelengths (νP and νS, the pump laser and Stokes laser) are usually used for CARS spectroscopy; νS should be tunable in order to generate a CARS spectrum νCARS (νCARS=2νp−νS, ICARS˜(IP)2·IS). FIG. 2 schematic energy-level diagram of a CARS transition. If the frequency difference νP −νSmatches the frequency difference between two molecular vibration states |1> and |0> in the sample, the CARS signal is then in fact amplified further. In microscope applications, the pump light beam and Stokes light beam are combined coaxially and focused together onto the same sample volume. The direction in which the anti-Stokes radiation is emitted is determined by the phase adaptation condition for the four-wave mixing process, as depicted schematically in FIG. 3.
U.S. Pat. No. 4,405,237 “Coherent anti-Stokes Raman device” discloses an apparatus in which two pulsed laser beams, generated by two lasers and having different wavelengths in the visible region or the UV region of the spectrum, are used to illuminate a sample simultaneously. With suitably selected wavelengths, the sample can be excited in such a way that it emits the characteristic coherent anti-Stokes Raman radiation.
U.S. Pat. No. 5,194,912 “Raman analysis apparatus” discloses a microscope that contains an adjustable interference filter in the detection beam path. The interference filter is adjustable in such a way that the portion of the detection light having the desired Raman lines arrives at the detector.
U.S. Pat. No. 6,108,081 “Nonlinear vibrational microscopy” discloses a method and an apparatus for microscopic CARS spectroscopy. This document discloses generation of a pump light beam with a titanium-sapphire laser, and a Stokes light beam with an optically parametric oscillator, which are combined into a coaxial illuminating light beam using a dichroic beam combiner. A regenerative amplifier is additionally provided in order to achieve sufficiently high pulse power levels.
Distinct disadvantages of the existing technology result from the limitations of the excitation light source, which usually is very complex because it comprises a pulsed laser (usually a titanium-sapphire laser), additionally a regenerative amplifier that makes low repetition rates (in the kHz range) unavoidable, and furthermore a complex and expensive optically parametric oscillator (OPO) that is pumped by a sub-beam of the pulsed laser. The known illumination arrangements are complex. expensive, and difficult to align; in particular, coaxial superimposition of the pump light beam and Stokes light beam requires a special alignment effort in order to ensure that the two beams are focused onto the same sample volume. The beam splitter used in the existing art for beam combination is usable only for specific combinations of pump light beam wavelength and Stokes light beam wavelength, representing a further disadvantage. If different wavelength combinations are selected, the beam splitter must be replaced—a long and laborious task.
A further disadvantage of the known apparatuses is the small available wavelength region, which also limits the range of samples that can be investigated.